Particularly for machines producing liquid packaging board, or other food-grade packaging boards, a low bacterial spore content in the final product is an important quality factor. Spore forming bacterial cells are normally present in the environment of the board machine either as vegetative cells multiplying by cell division (vegetative form), or as a spore-form very resistant to harsh environmental conditions (dormant form surviving for years). Transformation of bacterial cells from the vegetative form into the resistant dormant form is called sporulation, whereas the retransformation of bacterial spore to give vegetative cell is called germination. In some board machines, low spore contents of the final product are difficult to achieve due to excessive bacterial sporulation. Eradication of mature spores requires high biocide concentrations, and accordingly, the prevention of the bacterial cells from sporulating would be more efficient in comparison to killing of mature spores. In case the bacterial sporulation is prevented, the cells in the process remain in the vegetative state, thus being more sensitive to biocides and killed by high temperatures of the drying section at the latest.
Factors influencing bacterial cell sporulation and germination are widely studied. It is known that sporulation is a relatively strictly controlled process that may not be stopped once initiated. Moreover, cells only sporulate under environmental stress, for instance when starving. Recently González-Pastor et al. (2003) showed that in a shortage of nutrients Bacillus subtilis bacteria, first try to delay sporulation by cannibalism. So-called skf (sporulation killing factor) and sdp (sporulation delay protein) genes are activated in cells starving first, causing secretion of cytotoxic agents and death of the surrounding sister cells. Nutrients released from the dead cells were utilized by the surviving ones. Fujita et al. (2005) showed that the initiation of sporulation depends on the so-called Spo0A control unit controlling the expression of about 121 genes involved in sporulation. Increase of the Spo0A protein first resulted in the control of genes supporting growth, thus causing e.g. killing of sister cells, utilization of released nutrients and increased biofilm formation, the actual sporulation process proceeding only later.
There are several studies directed to the resistance of mature spores to environmental stress factors. Turner et al. (2000) have studied the influence of antimicrobial agents on mature spores of the Bacillus subtilis bacteria. Efficiency of biocides was reduced as the cortex, inner and outer coats of the spores were finished during progressing sporulation. In the early stages of sporulation, the spores were resistant to toluene, formaldehyde, phenol, and phenylmercuric nitrate. Once the spore cortex was ready, the spores were resistant to chlorohexidine diacetate (CHA), quaternary ammonium compounds (QAC), and compounds releasing chlorine. In the final stage of sporulation, once the inner and outer spore coats were finished, resistances to the lysozyme enzyme and glutaraldehyde were also found. Resistance to biocides of mature spores is considerably greater than that of bacterial cells in the vegetative state. Finished board may not contain too high residual amounts of biocides, and accordingly, applied biocide amounts are limited for several board machines, thus preventing in practice the use of biocides in amounts necessary for the eradication of mature spores. Heat-resistant spores also survive the high temperatures of the drying section of the board machine, which are normally lethal to vegetative cells.
Several metal ions including manganese are involved in the growth, sporulation and germination of microbes. Several studies about the influence of various metals on different enzymatic activities may be found in the literature. Charney et al. (1951) showed that manganese is an important transition metal in the sporulation of the Bacillus subtilis bacteria. In this study, no sporulation of the bacterium took place in a growth medium very rich in nutrients but having a low manganese concentration. The amount of sporulating cells was increased by the manganese addition of 0.1 ppm or more. Vasantha and Freese (1979) examined the role of manganese in the growth and sporulation of the Bacillus subtilis bacteria. Manganese was shown to be an important metal for the activity of the phosphoglycerate phosphomutase enzyme during sporulation. Sporulation was successful in the absence of manganese only if glucose, malate and decoyinine inhibitor were added to the medium, said inhibitor preventing the formation of guanosine monophosphate synthase thus avoiding 3-phosphoglyceridic acid metabolic pathway. Accordingly, cells need manganese for normal sporulation pathway. Inaoka et al. (1999) showed that the SodA (superoxide dismutase) enzyme of the Bacillus subtilis bacteria in combination with manganese protected cells against external oxidants both in the growth and sporulation stages, that is, manganese is also an important cell protection factor. Que and Helmann (2000) showed that mnt genes of the Bacillus subtilis bacteria are involved in the transportation of manganese. Mutation of these genes prevented manganese transportation causing reduced sporulation in comparison to the sporulation of wild type strains. The amount of sporulating cells of the wild type strain was about 2.6% for a 0.006 ppm manganese addition, said amount being 39% in case 0.8 ppm of manganese was added. Accordingly, the addition of manganese clearly increased sporulation.
Metabolism of a bacterial spore is known to be rather minute. However, even in their dormant stage, spores have an influence on manganese present in their environment. Francis and Tebo (2002) showed that enzymes able to oxidize manganese from the soluble Mn(II) form to the insoluble Mn(IV) form may be isolated from the surface of the spores.
The fact that the growth of microbes may be limited by chelating iron and other metals has already been known for a long time, but however, chelates are typically not used for this purpose since necessary concentrations thereof are often very high. For instance in board machines, various biocides are used for microbial growth inhibition. Fortnagel and Freese (1968) have studied the influence of chelates on sporulation. They showed in a basic research directed to the mechanism of the sporulation of the Bacillus subtilis bacteria that sporulation could be stopped by chelators binding transition metals, thus inhibiting the aconitase enzyme. Growth was not inhibited by alpha picolinic acid concentrations of less than 1 mM (<123 ppm), whereas a concentration of 0.4 mM reduced sporulation. Aconitase enzyme was also inhibited by concentrations of less than 1 mM of quinaldic acid, o-phenanthroline, and dipyridyl.
In paper industry, chelation of manganese has been studied with respect to bleaching processes. The purpose of the study by Kujala et al. (2004) was the reduction of the amount of manganese naturally present in wood during pulping process since it decomposes hydrogen peroxide used for pulp bleaching. Manganese was best chelated by DTPMP, one of four chelating agents used in the study, i.e. NTA (nitrilotriacetic acid), EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid) and DTPMP (diethylenetriamine-pentakis methylenephosphonic acid), with a chelating performance of over 95%.